Syntheses, activities, and methods of use of dihydronicotinamide riboside derivatives

ABSTRACT

Disclosed is a compound of formula (I): 
                         
wherein R 1 , R 2 , R 3 , and are as defined herein. Also disclosed are methods for increasing mammalian cell NAD +  production and improving mitochondrial cell densities comprising administering to a cell the compound or a salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a Continuation of U.S. patent applicationSer. No. 15/744,602, filed Jan. 12, 2018, which is the U.S. nationalstage of International Patent Application No. PCT/US2016/042600, filedJul. 15, 2016, which claims the benefit of U.S. Provisional PatentApplication No. 62/192,917, filed Jul. 15, 2015, the disclosures ofwhich are incorporated in their entireties by reference for allpurposes.

BACKGROUND OF THE INVENTION

Nicotinamide riboside and derivatives thereof, including nicotinateriboside, nicotinamide mononucleotide, and nicotinate mononucleotide,are metabolites of nicotinamide adenine dinucleotide (NAD⁺). Currentwork available as state of the art establishes that increasing mammaliancell and tissue NAD⁺ production can provide a number of health benefits,these include but are not limited to protection from neurodegenerationcaused by Alzheimer's disease, resistance of mammals to toxic effects ofhigh fat diets, improvement in mitochondrial densities in animals,improvement in insulin sensitivity and improved exercise endurance.Other work has provided hints at protection from neurotrauma, such asblast injury and noise induced hearing loss by enhancing NAD⁺.Neurogenesis has also been linked to this effect. NAD⁺ enhancement bypharmacologic agents mimics the effects of low calorie diets andexercise on physiology, and is a low toxicity method for mimicking thebeneficial effects of these health beneficial regimes on humanphysiology. Moreover, the effect of increased physiologic NAD⁺ appearsto be to increased sirtuin activity, which is responsible for some ofthe beneficial effects observed for low calorie diets and exercise onhuman physiology.

While nicotinamide riboside and ester analogs thereof are useful asefficient precursors of NAD⁺ to elevate levels of NAD⁺ and thus promotecellular health, the bioavailability of these molecules may not beoptimal as pharmacological and nutritional agent. Accordingly, thereremains a need in the art for agents that elevate cellular levels ofNAD⁺ that possess suitable bioavailability and stability, and are freefrom adverse effects on NAD⁺-dependent biological systems.

BRIEF SUMMARY OF THE INVENTION

The invention provides a compound of formula (I):

wherein R¹, R², and R³ are independently selected from hydrogen, formyl,optionally substituted C₁-C₁₂ alkylcarbonyl, and optionally substitutedC₆-C₁₀ arylcarbonyl,

X is NHR⁴, NR⁴R⁵, or OR⁶,

R⁴ and R⁵ are independently optionally substituted C₁-C₁₂ alkyl oroptionally substituted C₆-C₁₀ aryl, and

R⁶ is hydrogen, optionally substituted C₁-C₁₂ alkyl, or optionallysubstituted C₆-C₁₀ aryl,

wherein the alkyl or aryl portion of R⁴-R⁶ is optionally substitutedwith one or more substituents selected from halo, amino, alkyl, alkoxy,aryloxy, hydroxyalkyl, and any combination thereof,

or a pharmaceutically acceptable salt thereof.

The invention also provides a process for the preparation of a compoundof formula (II):

wherein R¹, R², and R³ are independently selected from hydrogen, formyl,optionally substituted C₁-C₁₂ alkylcarbonyl, and optionally substitutedC₆-C₁₀ arylcarbonyl, and

R⁶ is hydrogen, optionally substituted C₁-C₁₂ alkyl, or optionallysubstituted C₆-C₁₀ aryl,

wherein the alkyl or aryl portion of R⁶ is optionally substituted withone or more substituents selected from halo, amino, alkyl, alkoxy,aryloxy, hydroxyalkyl, and any combination thereof,

wherein the process comprises the steps of:

(i) providing a compound of formula (III):

(ii) reducing the compound of formula (III) with a reducing agent toprovide the compound of formula (II), and

(iii) isolating the compound of formula (II).

The invention additionally provides a method for increasing cell NAD⁺production comprising administering to a cell a compound of formula (I)or a salt thereof.

The invention further provides a method of improving mitochondrialdensities in a cell, wherein the method comprises administering to thecell a compound of formula (I) or a salt thereof.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1A shows the viable cell counts for HEK293 cells treated with wateror dihydronicotinamide riboside.

FIG. 1B shows the viable cell counts for Neuro2A cells treated withwater or dihydronicotinamide riboside.

FIG. 1C shows the non-viable cell counts for HEK293 cells treated withwater or dihydronicotinamide riboside.

FIG. 1D shows the non-viable cell counts for Neuro2A cells treated withwater or dihydronicotinamide riboside.

FIG. 2A shows NAD⁺ levels resulting from a 16 hour treatment of HEK293cells with water, 100 μM NRH, 500 μM dihydronicotinamide riboside, and500 μM nicotinamide riboside.

FIG. 2B shows NAD⁺ levels resulting from an 8 hour treatment of HEK293cells with water, 100 μM dihydronicotinamide riboside, 500 μM, 1000 μMdihydronicotinamide riboside, and 1900 μM nicotinamide riboside.

FIG. 2C shows NAD⁺ levels resulting from a 16 hour treatment of HEK293cells with water, 750 μM dihydronicotinamide riboside, and 750 μMnicotinamide riboside.

FIG. 3A shows NAD⁺ levels resulting from treatment of Neuro2A cells withwater, 500 μM dihydronicotinamide riboside, and 500 μM nicotinamideriboside.

FIG. 3B shows NAD⁺ levels resulting from treatment of Neuro2A cells withDMSO, 500 μM dihydronicotinamide riboside, and 500 μM nicotinamideriboside.

FIG. 4 shows cell counts for Neuro2A cells treated with vehicle, 1000 μMdihydronicotinamide riboside, vehicle+hydrogen peroxide, and 1000 μMdihydronicotinamide riboside+hydrogen peroxide.

FIG. 5 shows the average NAD⁺ concentration in Nuero2A cells treatedwith water or dihydronicotinamide riboside at 0 h, 1 h, and 7 h.

FIG. 6 shows cellular NAD⁺ levels in Neuro2A cells treated withincreasing concentrations of dihydronicotinamide riboside.

FIG. 7 shows cellular NAD⁺ levels in INS1 cells treated with increasingconcentrations of dihydronicotinamide riboside.

FIG. 8 shows cellular NAD⁺ levels in primary neuron cells treated withincreasing concentrations of dihydronicotinamide riboside.

FIG. 9 shows the average concentration of NAD⁺ in the mitochondria andcytoplasm of Neuro2A cells treated with dihydronicotinamide riboside.

FIG. 10 shows the concentration over time of dihydronicotinamideriboside in aqueous solutions at pH values of 6, 7, 7.4, 8, and 9.

FIG. 11A shows the cell number and average NAD⁺ concentration forNeuro2A cells treated with dihydronicotinamide riboside, hydrogenperoxide, or dihydronicotinamide riboside+hydrogen peroxide.

FIG. 11B shows the cell number and average NAD⁺ concentration for INS1cells treated with dihydronicotinamide riboside, hydrogen peroxide, ordihydronicotinamide riboside+hydrogen peroxide.

FIG. 12A shows the cell number and average NAD⁺ concentration forNeuro2A cells treated with dihydronicotinamide riboside, methylmethanesulfonate (MMS), or dihydronicotinamide riboside+MMS.

FIG. 12B shows the cell number and average NAD⁺ concentration for HEK293cells treated with dihydronicotinamide riboside, methyl methanesulfonate(MMS), or dihydronicotinamide riboside+MMS.

FIG. 13 shows the cell number and average NAD⁺ concentration for HEK293cells treated with dihydronicotinamide riboside, vincristine (VIN), ordihydronicotinamide riboside+VIN.

FIG. 14A shows the average NAD⁺ concentration in INS1 cells treated with3 mM glucose and with control, dihydronicotinamide riboside, hydrogenperoxide, dihydronicotinamide riboside+hydrogen peroxide, andnicotinamide riboside+hydrogen peroxide in the presence of 20 nMglucose.

FIG. 14B shows the concentration of insulin in the media produced byINS1 cells treated with 3 mM glucose and with control,dihydronicotinamide riboside, hydrogen peroxide, dihydronicotinamideriboside+hydrogen peroxide, and nicotinamide riboside+hydrogen peroxidein the presence of 20 nM glucose.

FIG. 15 shows the average NAD⁺ concentration in HEK293 cells incubatedfor 6 h with the indicated concentrations of dihydronicotinamideriboside, 1,4-dihydro-ethyl nicotinate riboside, and 1,4-dihydro-propylnicotinate riboside.

FIG. 16 shows blood levels of NAD+ in C57/B6 mice injected i.p. with1000 mg/kg of dihydronicotinamide riboside at 2 h and 4 h afterinjection.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a compound of formula (I):

wherein R¹, R², and R³ are independently selected from hydrogen, formyl,optionally substituted C₁-C₁₂ alkylcarbonyl, and optionally substitutedC₆-C₁₀ arylcarbonyl,

X is NHR⁴, NR⁴R⁵, or OR⁶,

R⁴ and R⁵ are independently optionally substituted C₁-C₁₂ alkyl oroptionally substituted C₆-C₁₀ aryl, and

R⁶ is hydrogen, optionally substituted C₁-C₁₂ alkyl, or optionallysubstituted C₆-C₁₀ aryl,

wherein the alkyl or aryl portion of R⁴-R⁶ is optionally substitutedwith one or more substituents selected from halo, amino, alkyl, alkoxy,aryloxy, hydroxyalkyl, and any combination thereof,

or a pharmaceutically acceptable salt thereof.

In certain embodiments, when X is OR⁶ and R⁶ is hydrogen, R¹, R², and R³are not all hydrogen or acetyl.

In certain embodiments, X is OR⁶ and R⁶ is hydrogen, R¹ is not benzoyland R² and R³ are not hydrogen.

In certain embodiments, when X is OR⁶ and R⁶ is methyl or ethyl, R¹, R²,and R³ are not all acetyl.

In certain embodiments, X is OR⁶, R⁶ is optionally substituted C₁-C₁₂alkyl, and R¹, R², and R³ are hydrogen.

In particular embodiments, the compound is selected from:

In certain embodiments, X is OR⁶, R⁶ is optionally substituted C₁-C₁₂alkyl, and R¹, R², and R³ are C₁-C₁₂ alkylcarbonyl.

In certain embodiments, X is NHR⁴.

In certain embodiments, X is NR⁴R⁵.

In certain of the above embodiments, R¹, R², and R³ are hydrogen.

In certain of the above embodiments, R¹, R², and R³ are C₁-C₁₂alkylcarbonyl.

The phrase “salt” or “pharmaceutically acceptable salt” or “saltacceptable for use in dietary supplements or food ingredients” isintended to include nontoxic salts synthesized from the parent compoundwhich contains a basic or acidic moiety by conventional chemicalmethods. Generally, such salts can be prepared by reacting the free acidor base forms of these compounds with a stoichiometric amount of theappropriate base or acid in water or in an organic solvent, or in amixture of the two. Generally, nonaqueous media such as ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Company, Easton, Pa., 1990, p. 1445, and Journal ofPharmaceutical Science, 66, 2-19 (1977). For example, they can be a saltof an alkali metal (e.g., sodium or potassium), alkaline earth metal(e.g., calcium), or salt of ammonium or alkylammonium, for example,monoalkylammonium, dialkylammonium, trialkylammonium, ortetraalkylammonium.

Examples of salts for use in the present inventive compositions includethose derived from mineral acids, such as hydrochloric, hydrobromic,phosphoric, metaphosphoric, nitric and sulphuric acids, and organicacids, such as tartaric, acetic, citric, malic, lactic, fumaric,benzoic, glycolic, gluconic, succinic, maleic and arylsulfonic acids,for example, methanesulfonic, trifluoromethanesulfonic, benzenesulfonic,and p-toluenesulfonic, acids.

The invention further provides a pharmaceutical composition, a dietarysupplement composition, or a food ingredient composition, comprising acompound as described above in any of the embodiments and apharmaceutically acceptable carrier. The invention provides apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an effective amount, e.g., a therapeutically effectiveamount, including a prophylactically effective amount, of one or more ofthe aforesaid compounds, or salts thereof, of the invention. Theinvention also provides a dietary supplement or food ingredientcomposition comprising an acceptable carrier and an effective amount,e.g., an amount to affect the structure or function of the body, of oneor more of the aforesaid compounds, or salts thereof, of the invention.

The acceptable carrier can be any of those conventionally used and islimited only by chemico-physical considerations, such as solubility andlack of reactivity with the compound, and by the route ofadministration. It will be appreciated by one of skill in the art that,in addition to the following described pharmaceutical, dietarysupplement, or food ingredient compositions; the compounds of theinvention can be formulated as inclusion complexes, such as cyclodextrininclusion complexes, or liposomes.

The acceptable carriers described herein, for example, vehicles,adjuvants, excipients, or diluents, are well known to those who areskilled in the art and are readily available to the public. It ispreferred that the acceptable carrier be one which is chemically inertto the active compounds and one which has no detrimental side effects ortoxicity under the conditions of use.

The choice of carrier will be determined in part by the particularactive agent, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of the pharmaceutical, dietary supplement, and foodingredient compositions of the invention. The following formulations fororal, aerosol, parenteral, subcutaneous, intravenous, intraarterial,intramuscular, interperitoneal, intrathecal, rectal, and vaginaladministration are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptable (oracceptable for dietary supplements or food ingredients) surfactant,suspending agent, or emulsifying agent. Capsule forms can be of theordinary hard- or soft-shelled gelatin type containing, for example,surfactants, lubricants, and inert fillers, such as lactose, sucrose,calcium phosphate, and cornstarch. Tablet forms can include one or moreof lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to the active ingredient, such carriers as areknown in the art.

The compounds of the invention, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compound can be administered in a physiologically acceptable diluentin a pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol,glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers,such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acidester or glyceride, or an acetylated fatty acid glyceride with orwithout the addition of a pharmaceutically acceptable surfactant, suchas a soap or a detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyl dialkyl ammoniumhalides, and alkyl pyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene-polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazolinequaternary ammonium salts, and (3) mixtures thereof.

The parenteral formulations will typically contain from about 0.5 toabout 25% by weight of the active ingredient in solution. Suitablepreservatives and buffers can be used in such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5 to about15% by weight. Suitable surfactants include polyethylene sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The compounds of the invention may be made into injectable formulations.The requirements for effective pharmaceutical carriers for injectablecompositions are well known to those of ordinary skill in the art. SeePharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia,Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbookon Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).

Additionally, the compounds of the invention may be made intosuppositories by mixing with a variety of bases, such as emulsifyingbases or water-soluble bases. Formulations suitable for vaginaladministration may be presented as pessaries, tampons, creams, gels,pastes, foams, or spray formulas containing, in addition to the activeingredient, such carriers as are known in the art to be appropriate.

The invention also provides a dietary supplement or food ingredientcomposition comprising a compound as described above in any of theembodiments. The terms dietary supplement and food ingredient as usedherein denotes the usefulness in both the nutritional field ofapplication. The dietary supplement and food ingredient compositionsaccording to the invention may be in any form that is suitable foradministrating to the animal body including the human body, especiallyin any form that is conventional for oral administration, e.g. in solidform such as (additives/supplements for) food or feed, food or feedpremix, tablets, pills, granules, dragees, capsules, and effervescentformulations such as powders and tablets, or in liquid form such assolutions, emulsions or suspensions as e.g. beverages, pastes and oilysuspensions. Controlled (delayed) release formulations incorporating thecompounds according to the invention also form part of the invention.Furthermore, a multi-vitamin, mineral, or other supplement may be addedto the dietary supplement and food ingredient compositions of theinvention to obtain an adequate amount of an essential nutrient, whichis missing in some diets, or to further aid the structure or function ofthe body. The multi-vitamin and mineral supplement may also be usefulfor disease prevention and protection against nutritional losses anddeficiencies due to lifestyle patterns.

Chemistry

The compounds of the invention can prepared by any suitable process. Forexample, the process can comprise the steps of (i) providing a compoundof formula (IV):

wherein R¹, R², and R³ are independently selected from hydrogen, formyl,optionally substituted C₁-C₁₂ alkylcarbonyl, and C₆-C₁₀ optionallysubstituted arylcarbonyl, X is NHR⁴, NR⁴R⁵, or OR⁶, R⁴ and R⁵ areindependently optionally substituted C₁-C₁₂ alkyl or optionallysubstituted C₆-C₁₀ aryl, and R⁶ is hydrogen, optionally substitutedC₁-C₁₂ alkyl, or optionally substituted C₆-C₁₀ aryl,

(ii) treating the compound of formula (IV) with a reducing agent toprovide the compound of formula (V),

and (iii) isolating the compound of formula (V).

The compound of formula (IV) can be prepared using any suitable process.For example, the compound of formula (IV) can be prepared by processesdisclosed in U.S. Patent Application Publication 2007/0117765 A1, thecontents of which are totally incorporated herein by reference.

In an embodiment, the invention provides a process for the preparationof a compound of formula (II):

wherein R¹, R², and R³ are independently selected from hydrogen, formyl,optionally substituted C₁-C₁₂ alkylcarbonyl, and C₆-C₁₀ optionallysubstituted arylcarbonyl, and

R⁶ is hydrogen, optionally substituted C₁-C₁₂ alkyl, or optionallysubstituted C₆-C₁₀ aryl,

wherein the alkyl or aryl portion of R⁶ is optionally substituted withone or more substituents selected from halo, amino, alkyl, alkoxy,aryloxy, hydroxyalkyl, and any combination thereof,

wherein the process comprises the steps of:

(i) providing a compound of formula (III):

(ii) treating the compound of formula (III) with a reducing agent toprovide the compound of formula (II), and

(iii) isolating the compound of formula (II).

The reducing agent can be any suitable reducing agent and can be, forexample, sodium dithionate, sodium borohydride, hydrogen in the presenceof a catalyst, and the like.

Isolation of the compound of formula (II) can be accomplished using anysuitable isolation technique. In embodiments, the isolation is conductedby chromatography. Non-limiting examples of suitable chromatographictechniques include normal phase chromatography over silica gel or highperformance liquid chromatography (HPLC) over reversed phase supports.In some embodiments, the isolation can be conducted by crystallizationfrom a suitable solvent or mixture of solvents.

Exemplars of the compounds of the invention exhibit a surprising andunexpected effect on mammalian cells vis-a-vis NAD⁺ increases. Theseoccur at relatively low concentrations and increases in NAD⁺ in excessof 400% can be obtained at 20-200 microM where other compounds at anyconcentration are not efficacious to achieve the effect. Because ofreduction of the ring, the compounds are also more lipophilic than theirrespective aromatic relatives, and this may increase absorption and BBBpenetration characteristics. The synthetic methods themselves providethe first fully efficient methods to achieve the compounds. The keyfeatures of the compounds appear to be potency, ease of access, novelty,lack of literature characterization of the biological properties,evidence for biological efficacy in enhancing NAD⁺ above previouslydescribed molecules, and opportunities for improved drug behavior fromenhanced lipophilicity.

In some embodiments, the invention provides a method for increasingmammalian cell NAD⁺ production comprising administering to a cell acompound of the invention or a pharmaceutically acceptable salt thereof.Nicotinamide adenine dinucleotide (NAD or NAD⁺) is important as aco-enzyme for different enzymes. Recent studies depicted that being theco-substrate of SIR2 (silent information regulator 2), NAD⁺ has a rolein regulating multiple biological processes, such as p53 regulatedapoptosis, fat storage, stress resistance, and gene silencing. Withoutlimiting the potential uses of the compositions described herein by anysingle theory, there are various pathways through which nicotinamideriboside (NR), dihydronicotinamide riboside (NRH), nicotinic acidriboside (NAR), and dihydronicotinic acid riboside (NARH or NaR-H) andtheir derivatives are currently thought to be metabolized. NR and NRHare known as NAD⁺ precursors for both human and yeast. They are able toenter a salvage pathway that leads to biological synthesis of NAD⁺ underthe action of the enzyme nicotinamide riboside kinase (Nrk). Each of NRand NRH can be converted to nicotinamide mononucleotide (NMN) andnicotinic acid mononucleotide (NAMN) by nicotinamide riboside kinases(Nrk), which are then converted to NAD⁺ by the enzyme nicotinamidemononucleotide adenylytransferase (Nmnat). Alternatively, NR and NRH canenter NAD metabolism by means of other metabolic paths, which wouldinclude action from enzymes that separate the nicotinamide moiety fromthe sugar. Such a path would include the action of phosphorylases thathave been shown to degrade NR and NRH in cells to form nicotinamide andribose-1-phosphate. Nicotinamide is competent to enter NAD⁺ metabolismand is converted to NAD+ by the action of the enzyme nicotinamidepyrophosphoribosyltransferase. Sirtuins are class III histonedeacetylases (HDACs) and are ADP-ribosyl transferases also. Theydeacetylate lysine residues in a novel chemical reaction that consumesnicotinamide adenine dinucleotide (NAD⁺), releasing nicotinamide,O-acetyl-ADPribose (AADPR), and the deacetylated substrate. Alteringintracellular NAD⁺ levels can improve the health of a cell, butintroduction of compounds that enter NAD⁺ metabolic pathways can alsoprove toxic to cells. In some embodiments, the invention relates to theuse of compounds disclosed herein to manipulate NAD⁺ levels, to modulatethe activity of sirtuins and other ADP-ribosyl transferases, and tomodulate inosine 5′-monophosphate dehydrogenase. These embodiments areused to destroy or weaken the defenses of cancer cells, or to promotesurvival of neurons, myocytes, or stem cells via addition to growthmedia.

Nicotinic acid is an effective agent in controlling low-densitylipoprotein cholesterol, increasing high-density lipoproteincholesterol, and reducing triglyceride and lipoprotein (a) levels inhumans. Though nicotinic acid treatment affects all of the key lipids inthe desirable direction and has been shown to reduce mortality in targetpopulations, its use is limited because of a side effect of heat andredness termed flushing. Further, nicotinamide is neuroprotective inmodel systems, presumably due to multiple mechanisms includingincreasing mitochondrial NAD⁺ levels. NR and derivatives thereof hasalso proved useful in model systems and in clinical trials in humans ina variety of uses, including promoting healthy aging, supporting andpromoting healthy metabolic function, supporting and promoting cognitivefunction, neuroprotection in CNS and PNS trauma including stroke, and inneurogenerative diseases and conditions including essential tremor,Parkinson disease, Alzheimer disease, Huntington disease, ataxia,catatonia, epilepsy, neuroleptic malignant syndrome, dystonia,neuroacanthocytosis, Pelizaeus-Merzbacher, progressive supranuclearpalsy, Striatonigral degeneration, Tardive dyskinesias, or a lysosomalstorage disorder, including lipid storage disorders (including Gaucher'sand Niemann-Pick diseases), gangliosidosis (including Tay-Sachsdisease), leukodystrophies, mucopolysaccharidoses, glycoprotein storagedisorders, and mucolipidoses. They have also been found useful toprevent hearing loss due to aging or exposure to loud sounds. They alsocan protect cells from damage to exposure to toxins, including damage tomyocytes caused by statins. They can slow or prevent the death of isletcells that produce insulin. They have also been found to increase thenumber of, and improve the function of, mitochondria.

NRH or derivatives may be bioavailable and are ultimately convertible bymetabolism to nicotinic acid or nicotinamide and to NAD+, therebyproviding the benefits of the compounds as discussed above. Accordingly,one embodiment of the invention relates to the use of compositionscomprising compounds disclosed herein that work through the nicotinamideriboside kinase pathway or other pathways of NAD⁺ biosynthesis whichhave nutritional and/or therapeutic value in improving poor plasma lipidprofiles in lipid disorders, (e.g., dyslipidemia, hypercholesterolaemiaor hyperlipidemia), metabolic dysfunction in type I and II diabetes,cardiovascular disease, and other physical problems associated withobesity, protecting islet cells to treat or prevent development ofdiabetes, neuroprotection to treat trauma and neurodegenerative diseasesand conditions, protecting muscle cells from toxicity and damage fromworkouts or trauma, promoting the function of the auditory system,treating or preventing hearing loss, and dietary supplement and foodingredient uses for promoting metabolic function, muscle function andhealing/recovery, cognitive function, and mitochondrial function.

In some embodiments, the invention relates to the use of compoundsdisclosed herein as agonist and antagonist of enzymes in the pathway ofNAD⁺ biosynthesis. In further embodiments, the NHR derivatives disclosedherein are agonist, i.e., stimulates activities normally stimulated bynaturally occurring substances, of one or more sirtuins, preferablySIRT1 in humans or Sir2p in yeast. In further embodiments, the NHRderivatives are antagonist of one or sirtuins.

In some embodiments, the invention provides a method of improvingmetabolic function, including increased mitochondrial densities, insulinsensitivity, or exercise endurance in a mammal, wherein the methodcomprises administering to the mammal a compound of the invention or apharmaceutically acceptable salt, or salt acceptable for dietarysupplements or food ingredients, thereof. It is known that under calorierestriction, cellular energy depletion causes rising AMP levels, and anincrease in the NAD⁺ level as compared to the reduced level (NADH),results in activation of AMPK. AMPK activation leads to PGC-1alphaactivation which leads to mitochondrial biosynthesis (Lopez-Lluch, etal., Experimental Gerontology, 2009 September, 43 (9): 813-819doi:10.1016/j.exger.2008.06.014). Increasing mitochondrial biosynthesiswill lead to increased mitochondrial density in the muscle cells.Increased mitochondrial density will increase athletic performance interms of muscle strength and endurance.

In some embodiments, the invention provides a method of treating orpreventing a disease or condition in a mammal in need thereof, whereinthe method comprises administering to the mammal a compound of theinvention or a pharmaceutically acceptable salt thereof, wherein thedisease or condition is CNS or PNS trauma, or a neurodegenerativedisease or condition.

NAD⁺ levels decrease in injured, diseased, or degenerating neural cellsand preventing this NAD⁺ decline efficiently protects neural cells fromcell death. Araki & Milbrandt, Science, 2004 Aug. 13; 305(5686):1010-3and Wang et al., “A local mechanism mediates NAD-dependent protection ofaxon degeneration,” J. Cell Biol. 170(3): 349-55 (2005), herebyincorporated by reference. As a number of inventive compounds disclosedherein are capable of increasing intracellular levels of NAD⁺, thesecompounds are useful as a therapeutic or nutritional supplement inmanaging injuries, diseases, and disorders effecting the central nervoussystem and the peripheral nervous system, including but not limited totrauma or injury to neural cells, diseases or conditions that harmneural cells, and neurodegenerative diseases or syndromes. Someneurodegenerative diseases, neurodegenerative syndromes, diseases andconditions that harm neural cells, and injury to neural cells aredescribed above. It is preferred that inventive compounds disclosedherein are capable of passing the blood-brain-barrier (BBB).

In some embodiments, the invention provides a method of protecting amammal from neurotrauma, wherein the method comprises administering tothe mammal a compound of the invention or a pharmaceutically acceptablesalt thereof. In certain of these embodiments, the neurotrauma resultsfrom blast injury or noise. In these embodiments, the agent increasesintracellular NAD⁺ in one or more cells selected from the groupconsisting of spiral ganglia nerve cells, hair cells, supporting cells,and Schwann cells.

In certain embodiments, the agent suppresses oxidative damage in thecell. In certain embodiments, the compound activates SIRT3. EndogenousSIRT3 is a soluble protein located in the mitochondrial matrix.Overexpression of SIRT3 in cultured cells increases respiration anddecreases the production of reactive oxygen species. Without wishing tobe bound by any particular theory, it is believed that activation ofSIRT3 is implicated in suppression of oxidative damage in the aforesaidcells.

In certain embodiments, the treating of the mammal with the compoundresults in prevention of hearing loss. In other embodiments, thetreating of the mammal with the agent results in the mitigation ofhearing loss. The treating can be performed after exposure to the mammalto circumstances leading to hearing loss, such as exposure to noise, orcan be performed prior to exposure of the mammal to the circumstances.The relationship of NAD⁺ levels and protection from neurotrauma isdisclosed in WO 2014/014828 A1, the contents of which are incorporatedherein by reference. In certain embodiments, the compound supports thehealthy structure or function of the auditory system in a mammal in needthereof. Treating of the mammal with an effective amount of thecompound, for example, in a dietary supplement or in a food ingredientcomposition, augments intracellular NAD⁺ biosynthesis, whereinintracellular NAD⁺ increases in spiral ganglia nerve cells, hair cells,supporting cells, Schwann cells, or a combination thereof. In someembodiments, the agent maintains axonal NAD+ levels following axonalinjuries caused by acoustic trauma.

Statins, more mechanistically known as 3-hydroxy-3-methyglutarylcoenzyme A reductase inhibitors (or HMG-CoA inhibitors), are some of theworld's most widely prescribed drugs. While statins are well toleratedat therapeutic doses, at higher doses and often in combination withother hypolipidaemic agents some potentially severe adverse effects havearisen. Most notably, cerivastatin (Baycol) was removed from the marketin 2000 after 31 deaths in the United States from drug-associatedrhabdomyolysis (breakdown of muscle fibers resulting in the release ofmuscle fiber contents into the circulation; some of these are toxic tothe kidney) and associated acute renal failure in patients takingcerivastatin. Statins are also known to have severe interactions withfibric acid derivatives, especially with gemfibrozil. Of the 31 peoplewho died taking cerivastatin, 12 were also taking gemfibrozil.

The most serious adverse effects of statins appear to occur in liver andmuscle cells, although it could be predicted that because of theirlipophilicity, cerebral effects might also be seen in some patients.

The exact mechanism of statin toxicities is unknown. The fact thattoxicities are dose-dependent makes plausible the hypothesis thattoxicities result from exaggeration of the drug's intended effect: inother words, cells die from lack of the downstream products of HMG-CoA.

HMG-CoA is the rate limiting enzyme in the mevalonate pathway, which,through three branches, leads to the synthesis of cholesterol, dolichol(the precursor to dolichol pyrophosphate, which is the first thing addedto proteins in post-translational glycosylation), and to ubiquinone,also known as Coenzyme Q (found in the membranes of endoplasmicreticulum, peroxisomes, lysosomes, vesicles and notably the innermembrane of the mitochondrion where it is an important part of theelectron transport chain; it is also has important antioxidantactivities).

However, it is likely that depletion of CoQ leads to a breakdown in theelectron transport chain, leading in turn to a buildup in NADH, and adepletion in NAD⁺. Further, the reduced form of CoQ10, CoQ10H2, has animportant cellular antioxidant function, which is to protect membranesand plasma lipoproteins against free radical-induced oxidation.

In some embodiments, the invention provides a method of reducingtoxicity induced by a HMGCoA reductase inhibitor in a mammal, whichmethod comprises administering to the mammal a therapeutically effectiveamount of a compound of the invention, wherein the mammal has beenadministered the HMGCoA reductase inhibitor in an amount that producestoxicity in the mammal in the absence of the administration of thecompound of formula (I), and wherein the administration of the compoundof claim 1 reduces the toxicity in the mammal. In some embodiments, theinvention provides a method of reducing toxicity induced by a HMGCoAreductase inhibitor in a mammal, which method comprises administering tothe mammal a therapeutically effective amount of a compound of theinvention and then administering to the mammal the HMGCoA reductaseinhibitor in an amount that produces toxicity in the mammal in theabsence of the administration of the compound of formula (I), wherebytoxicity that would have been induced by the HMGCoA reductase inhibitoris reduced in the mammal by the administration of the compound of theinvention. In some embodiments, the invention provides a method ofreducing toxicity induced by a HMGCoA reductase inhibitor in a mammal,which method comprises administering to the mammal a therapeuticallyeffective amount of a compound of the invention, whereby toxicityinduced by the HMGCoA inhibitor is reduced in the mammal, wherein thecompound of the invention is administered to the mammal followingmanifestation of toxicity by the mammal.

In some embodiments, the invention provides a method of reducingtoxicity induced by a genotoxic agent in a mammal, which methodcomprises administering to the mammal a therapeutically effective amountof a compound of the invention, wherein the mammal has been administeredthe genotoxic agent in an amount that produces toxicity in the mammal inthe absence of the administration of the compound of the invention, andwherein the administration of the compound reduces the toxicity in themammal. The compound of the invention can be administered to the mammalprior to administration of the genotoxic or other toxic agent to themammal, simultaneously with administration of the genotoxic or othertoxic agent to the mammal, or after administration of the genotoxic orother toxic agent to the mammal, for example, after symptoms of toxicityresulting from administration of the genotoxic or other toxic agentappear in the mammal.

In some embodiments, the invention relates to the use of a compound ofthe invention to prevent adverse effects and protect cells fromtoxicity. Toxicity may be an adverse effect of radiation or externalchemicals on the cells of the body. Examples of toxins arepharmaceuticals, drugs of abuse, and radiation, such as UV or X-raylight. Both radiative and chemical toxins have the potential to damagebiological molecules such as DNA. This damage typically occurs bychemical reaction of the exogenous agent or its metabolites withbiological molecules, or indirectly through stimulated production ofreactive oxygen species (eg, superoxide, peroxides, hydroxyl radicals).Repair systems in the cell excise and repair damage caused by toxins.

Enzymes that use NAD⁺ play an part in the DNA repair process.Specifically, the poly(ADP-ribose) polymerases (PARPs), particularlyPARP-1, are activated by DNA strand breaks and affect DNA repair. ThePARPs consume NAD⁺ as an adenosine diphosphate ribose (ADPR) donor andsynthesize poly(ADP-ribose) onto nuclear proteins such as histones andPARP itself. Although PARP activities facilitate DNA repair,overactivation of PARP can cause significant depletion of cellular NAD⁺,leading to cellular necrosis. The apparent sensitivity of NAD⁺metabolism to genotoxicity has led to pharmacological investigationsinto the inhibition of PARP as a means to improve cell survival.Numerous reports have shown that PARP inhibition increases NAD+concentrations in cells subject to genotoxicity, with a resultingdecrease in cellular necrosis. Nevertheless, cell death from toxicitystill occurs, presumably because cells are able to complete apoptoticpathways that are activated by genotoxicity. Thus, significant celldeath is still a consequence of DNA/macromolecule damage, even withinhibition of PARP. This consequence suggests that improvement of NAD⁺metabolism in genotoxicity can be partially effective in improving cellsurvival but that other players that modulate apoptotic sensitivity,such as sirtuins, may also play important roles in cell responses togenotoxins.

Physiological and biochemical mechanisms that determine the effects ofchemical and radiation toxicity in tissues are complex, and evidenceindicates that NAD⁺ metabolism is an important player in cell stressresponse pathways. For example, upregulation of NAD⁺ metabolism, vianicotinamide/nicotinic acid mononucleotide (NMNAT) overexpression, hasbeen shown to protect against neuron axonal degeneration, andnicotinamide used pharmacologically has been recently shown to provideneuron protection in a model of fetal alcohol syndrome and fetalischemia. Such protective effects could be attributable to upregulatedNAD⁺ biosynthesis, which increases the available NAD⁺ pool subject todepletion during genotoxic stress. This depletion of NAD⁺ is mediated byPARP enzymes, which are activated by DNA damage and can deplete cellularNAD⁺, leading to necrotic death. Another mechanism of enhanced cellprotection that could act in concert with upregulated NAD⁺ biosynthesisis the activation of cell protection transcriptional programs regulatedby sirtuin enzymes.

Examples of cell and tissue protection linked to NAD⁺ and sirtuinsinclude the finding that SIRT1 is required for neuroprotectionassociated with trauma and genotoxicity. SIRT1 can also decreasemicroglia-dependent toxicity of amyloid-beta through reduced NFKBsignaling. SIRT1 and increased NAD⁺ concentrations provideneuroprotection in a model of Alzheimer's disease. Sirtuins areNAD⁺-dependent enzymes that have protein deacetylase andADP-ribosyltransferase activities that upregulate stress responsepathways. Evidence indicates that SIRT1 is upregulated by calorierestriction and in humans could provide cells with protection againstapoptosis via downregulation of p53 and Ku70 functions. In addition,SIRT1 upregulates FOXO-dependent transcription of proteins involved inreactive oxygen species (ROS) detoxification, such as MnSOD. The sirtuinSIRT6 has been shown to participate in DNA repair pathways and to helpmaintain genome stability.

Pharmacological agents that target both NAD⁺ metabolism and sirtuins canprovide tools to elucidate the involvement of these factors inmodulating toxicity-induced tissue damage. Moreover, therapeutic optionsfor treatment of acute and chronic tissue-degenerative conditions canemerge if sirtuins and NAD⁺ metabolism can be validated as providingenhanced tissue protection. Agents such as the plant polyphenols (eg,resveratrol), the niacin vitamins, and the compound nicotinamideriboside can enhance cell survival outcomes by increasing NAD⁺biosynthesis, reducing NAD⁺ depletion, and/or activating sirtuinenzymes.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates a synthesis of1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-dihydropyridine-3-carboxamide,in accordance with an embodiment of the invention.

In a flame dried flask under an argon atmosphere, nicotinamide riboside(NR) (100 mg, 0.24 mmol) was added to 10 ml of 50 mM potassium phosphatedibasic (pH=8) at 0° C. After 5 minutes, 0.7 equivalents of sodiumdithionate (Na₂S₂O₄) were added and then reaction was run at 0° C. for30 minutes. The progress of the reaction was monitored by HPLC: 70% ofstarting material was consumed after 30 minutes. The crude product waspurified by chromatography over a C-18 column using water as eluent toobtain the title compound as a light yellow solid. Yield 70%.

¹H NMR (CD₃OD, 500 MHz): δ 7.08 (s, 1H, H-2), 6.02 (d, 1H, J=8.1 Hz,H-6), 4.94-4.89 (m, 1H, H-1′), 4.80 (d, 1H, J=7.1 Hz, H-5), 4.12 (t, 1H,J=6.3 Hz, H-3′), 4.06 (m, 1H, H-4′), 3.90-3.87 (m, 1H, H-2′), 3.69-3.58(m, 2H, H-5′), 2.99 (s, 2H, H-4a, H-4b). ¹³C NMR (CD₃OD, 125 MHz):137.8, 125.2, 105.2, 101.1, 94.9, 83.5, 70.9, 70.1, 61.5, 22.0.

Example 2

(This example demonstrates a synthesis of 2R,3S,4S,5R)-2-(acetoxymethyl)-5-(3-(ethoxycarbonyl)pyridin-1(4H)-yl)tetrahydrofuran-3,4-diyl diacetate, in accordance with anembodiment of the invention.

In a flame dried flask under an argon atmosphere, ethyl nicotinateriboside triacetate (200 mg, 0.5 mmol) was added to 20 ml of 50 m Mpotassium phosphate dibasic (pH=8) at 0° C. After 5 minutes, 0.7equivalents of sodium dithionate (Na₂S₂O₄) were added and the reactionwas run at 0° C. for 30 minutes. The progress of the reaction wasmonitored by HPLC: 60% of starting material was consumed after 30minutes. The crude product was purified by silica-gel column using ethylacetate:hexane (3:7) as eluent to obtain the title compound as a lightyellow solid. Yield 60%.

¹H NMR (CD₃OD, 500 MHz): δ 7.28 (s, 1H), 6.05 (dd, 1H, J=1.8 and 9.0Hz), 5.50 (s, 1H), 5.28-5.25 (m, 1H), 5.22 (t, 1H, J=7.0 Hz), 5.08 (1H,d, J=7.1 Hz), 4.94-4.89 (m, 1H), 4.26-4.24 (m, 1H), 4.20-4.10 (m, 3H),3.06 (s, 2H), 2.16 (s, 3H). 2.13 (s, 3H), 2.10 (s, 6H): ¹³C NMR (CD₃OD,125 MHz): 126.0, 105.2, 100.1, 92.6, 78.6, 70.5, 70.4, 63.8, 60.2, 59.7,22.6, 21.0, 20.9, 20.7, 14.9.

Example 3

This example demonstrates a synthesis ofethyl-1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4dihydropyridine-3-carboxylate,in accordance with an embodiment of the invention.

Ethyl nicotinate riboside (200 mg, 0.62 mmol) was added to 25 ml of 50mM potassium phosphate dibasic (pH=8) at 0° C. After 5 minutes, 75 mg ofsodium dithionate (Na₂S₂O₄, 0.7 equivalents, 0.43 mmol) were added andthe reaction was run at 0° C. for 30 minutes. The progress of thereaction was monitored by HPLC: 70% of starting material was consumedafter 30 minutes). The crude product was purified by C-18 column usingwater as eluent to provide the title compound as a light yellow solid.Yield 65%.

¹H NMR (CD₃OD, 500 MHz): δ 7.24 (s, 1H, H-2), 5.99 (d, 1H, J=6.9 Hz,H-6), 4.94-4.90 (m, 1H, H-1′), 4.78 (d, 1H, J=6.9 Hz, H-5), 4.14-4.03(m, 3H), 3.90-3.87 (m, 1H, H-4′), 3.69-3.58 (m, 2H, H2′), 3.26 (s, 1H,H-5′), 2.94 (s, 2H, H-4a, H-4b), 1.17 (t, 3H, —CH3, J=7.0 Hz); ¹³C NMR(CD₃OD, 125 MHz): 140.9, 124.5, 106.4, 99.2, 94.9, 83.6, 71.0, 70.2,61.4, 61.0, 21.6, 13.

Example 4

This example demonstrates a synthesis of butyl1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4dihydropyridine-3-carboxylate,in accordance with an embodiment of the invention.

Butyl nicotinate riboside nucleotide (50 mg, 0.10 mmol) was added to 4ml of 50 mM potassium phosphate dibasic (pH=8) at 0° C. After 5 minutes,19 mg of sodium dithionate (Na₂S₂O₄, 1 eq and 0.10 mmol) was added andthe reaction was run at 0° C. for 30 minutes. The progress of thereaction was monitored by TLC: 15% of starting material was consumedafter 30 minutes). The water layer was extracted with ethyl acetate. Thecrude product was purified by silica column using ethyl acetate aseluent to obtain the title compound as a solid. Yield 15%.

¹H NMR (CD₃OD, 500 MHz): δ 7.30 (s, 1H), 6.13 (d, 1H, J=8.5 Hz), 4.76(d, 1H, J=7.0 Hz), 4.11 (t, 2H, J=6.9 and 13.8 Hz), 4.07-4.01 (m, 2H),3.89-3.86 (m, 1H), 3.73-3.62 (m, 3H), 3.07 (s, 2H), 1.68-1.61 (m, 2H),1.47-1.39 (m, 2H), 0.97 (t, 3H. J=7.5 and 14.4 Hz). ¹³C NMR (CD₃OD, 125MHz): 169.0, 152.6, 149.7, 140.2, 137.5, 125.2, 123.9, 104.6, 98.2,95.9, 84.3, 72.0, 70.7, 65.1, 63.4, 61.9, 30.7, 22.1, 18.9, 12.7.

Example 5

This example demonstrates a synthesis of1-(1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-dihydropyridin-3-yl)octan-1-one,in accordance with an embodiment of the invention.

Heptyl nicotinate riboside (50 mg, 0.10 mmol) was added to 4 ml of 50 mMpotassium phosphate dibasic (pH=8) at 0° C. After 5 minutes, 18 mg ofsodium dithionate (Na₂S₂O₄, 1 equivalent, 0.10 mmol) was added and thereaction was run at 0° C. for 30 minutes. The progress of the reactionwas monitored by TLC: 15% of starting material was consumed after 30minutes). The water layer was extracted with 15 ml ethyl acetate. Theethyl acetate layer was concentrated and the crude product was purifiedby silica column using ethyl acetate as eluent to obtain the titlecompound as a solid. Yield 15%.

¹H NMR (CD₃OD, 500 MHz): δ 7.99 (s, 1H), 6.13 (d, 1H, J=8.3 Hz), 4.76(d, 1H, J=6.6 Hz), 4.10 (t, 2H, J=6.3 and 13.2 Hz), 4.07-4.02 (m, 2H),3.89-3.86 (m, 1H), 3.73-3.62 (m, 2H), 3.08-3.05 (m, 2H), 1.70-1.63 (m,2H), 1.43-1.29 (m, 9H), 0.95-0.89 (m, 3H). ¹³C NMR (CD₃OD, 125 MHz):146.6, 143.6, 140.2, 128.0, 125.1, 104.6, 101.3, 98.2, 95.9, 89.9, 84.3,78.7, 72.2, 70.9, 70.7, 66.9, 66.8, 65.0, 63.7, 61.9, 31.5, 28.7, 28.3,25.7, 25.5, 22.3, 22.1, 13.0.

Example 6

This example demonstrates a synthesis of1-(1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-dihydropyridin-3-yl)nonan-1-one,in accordance with an embodiment of the invention.

Octyl nicotinate riboside (50 mg, 0.10 mmol) was added to 4 ml of 50 mMpotassium phosphate dibasic (pH=8) at 0° C. After 5 minutes, 20 mg ofsodium dithionate (Na₂S₂O₄, 1 equivalent and 0.10 mmol) was added andthe reaction was run at 0° C. for 30 minutes. The progress of thereaction was monitored by TLC: 1 5% of starting material was consumedafter 30 minutes). The water layer was extracted with ethyl acetate. Thecrude product was purified by silica column using ethyl acetate aseluent to obtain the title compound as a solid. Yield 15%.

¹H NMR (CD₃OD, 500 MHz): δ 7.29 (s, 1H), 6.13 (d, 1H, J=8.4 Hz), 4.76(d, 1H, J=6.0 Hz), 4.10 (t, 2H, J=5.4 and 11.7 Hz), 4.07-4.02 (m, 2H),3.89-3.86 (m, 1H), 3.73-3.62 (m, 2H), 3.34-3.31 (m, 2H), 3.08-3.05 (m,2H), 1.69-1.62 (m, 2H), 1.46-1.03 (m, 8H), 0.98-0.90 (m, 4H). ¹³C NMR(CD₃OD, 125 MHz): 169.0, 140.2, 125.1, 104.6, 98.2, 95.9, 89.9, 78.7,72.0, 71.0, 70.7, 66.9, 66.8, 65.0, 63.7, 61.9, 61.6, 60.5, 32.3, 31.6,29.2, 29.1, 29.0, 28.6, 25.8, 25.6, 22.3, 22.1, 13.0.

Example 7

This example demonstrates a synthesis ofmethyl-1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-dihydropyridine-3-carboxylate,in accordance with an embodiment of the invention.

Methyl nicotinate riboside (200 mg, 0.47 mmol) was added to 20 ml of 50m M potassium phosphate dibasic (pH=8) at 0° C. After 5 minutes, 57 mgof sodium dithionate (Na₂S₂O₄, 0.7 equivalent, 0.32 mmol) was added andthe reaction was run at 0° C. for 30 minutes. The progress of thereaction was monitored by HPLC: 15% of starting material was consumedafter 30 minutes. The crude product was purified by silica column usingethyl acetate as eluent to obtain the title compound as a solid. Yield15%.

¹H NMR (CD₃OD, 500 MHz): δ 7.27 (s, 1H, H-2), 6.00 (d, 1H, J=7.5 Hz,H-6), 4.95-4.92 (m, 1H, H-1′), 4.80-4.78 (m, 1H, H-5), 4.12 (t, 1H,J=5.8 and 12.9 Hz, H-3′), 4.06-4.04 (m, 1H, H-4′), 3.98 (m, 1H, H-2′),3.91-3.87 (m, 2H, H-5′), 3.64-3.62 (s, 3H, OCH3).

Example 7

This example demonstrates a synthesis of isopentyl1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)1,4-dihydropyridine-3-carboxylate,in accordance with an embodiment of the invention.

Isoamyl nicotinate riboside (50 mg, 0.10 mmol) was added to 4 ml of 50mM potassium phosphate dibasic (pH=8) at 0° C. After 5 minutes, 20 mg ofsodium dithionate (Na₂S₂O₄, 1 equivalent, 0.10 mmol) was added and thereaction was run at 0° C. for 30 minutes. The progress of the reactionwas monitored by TLC: 15% of starting material was consumed after 30minutes. The water layer was extracted with 15 ml ethyl acetate. Thecrude product was purified by silica column using ethyl acetate aseluent to obtain the title compound as a solid. Yield 15%.

¹H NMR (CD₃OD, 500 MHz): δ 7.29 (s, 1H), 6.13 (d, 1H, J=6.3 Hz), 4.75(d, 1H, J=6.3 Hz), 4.43 (t, 1H, J=7.0 and 15.5 Hz), 4.14 (t, 2H, J=6.0and 14.4 Hz), 4.07-3.99 (m, 1H), 3.89-3.85 (m, 1H), 3.73-3.62 (m, 2H),3.06 (s, 2H), 1.59-1.53 (m, 2H), 1.05-0.99 (m, 2H), 0.98-0.91 (m, 6H).

Example 8

Methods

Cell Culture

HEK293 and Neuro2A cells were cultured in Dulbecco's modified Eagle'smedium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and100 μg/ml streptomycin. INS1 cells were maintained in RPMI-1640 with11.1 mmol/l D-glucose supplemented with 10% fetal bovine serum, 100 U/mlpenicillin and 100 μg/ml streptomycin, 10 mmol/l HEPES, 2 mmol/lL-glutamine, 1 mmol/l sodium pyruvate, and 50 μmol/l 2-mercaptoethanol.Cells were maintained in a humidified incubator supplied with 5% CO2/95%air at 37° C.

NRH Treatment for NAD⁺ Measurement

For NAD⁺ measurement, cells were seeded in 6-well plate for overnight.Then they were treated with desired concentration of NRH fromconcentrated stock dissolved in water and harvested with trypsindigestion after treatment time. Cell number were counted usinghemocytometer. The harvested cells were pelleted at 3000x g for 3 min.After removing the remaining media, cells were lysed with 7% perchloricacid to preserve NAD+, then neutralized with 2M NaOH and 500 mM K₂HPO₄.The cellular NAD⁺ level was measured using as previously published byour lab.

HEK Treated with Reduced NaR Esters.

HEK cells treated with reduced NaR esters (50 μM, 100 μM and 500 μM),Propyl NaR-H (50 μM, 100 μM and 500 μM) and NRH (50 μM and 500 μM) aspositive controls. After 6 hours of incubation, cells were harvestedwith Trypsin. A cell count was obtained from each cell suspension byhemacytometer. The cell pellets were used for NAD⁺ measurement. Theresults clearly show that NaR esters increase NAD concentrations.

Glucose-Induced Insulin Secretion Test in INS1 Cells

To induce insulin secretion, INS1 cells were seeded in 6-well plate. Thecells were accommodated to low glucose RPMI-1640 media with 5 mmol/lD-glucose overnight. Insulin secretion was assayed in HEPES balancedsalt solution (HBSS) (114 mmol/l NaCl, 4.7 mmol/l KCl, 1.2 mmol/lKH₂PO₄, 1.16 mmol/l MgSO₄, 20 mmol/l HEPES, 2.5 mmol/l CaCl₂, 25.5mmol/l NaHCO₃, and 0.2% bovine serum albumin [essentially fatty acidfree], pH 7.2). Cells were washed in 1 ml HBSS with 3 mmol/l glucosefollowed by a 2 hr preincubation in 2 ml of the same buffer. Insulinsecretion was then measured by incubating 1 hr 1 ml of HBSS containingthe 3 mmol/l or 15 mmol/l glucose concentration. The media was thencollected for insulin analysis using RayBio™ Rat Insulin ELISA kitaccording to manufacturer's manual. In hydrogen peroxide challenge, 100μM hydrogen peroxide were added to media together with 3 mmol/l glucosecontaining HBSS and incubated for 2 hr before switch to 15 mmol/lglucose containing HBSS for insulin secretion. 1 mM NRH or NR has beenadded to low glucose RPMI-1640 media for overnight and replenished whencells were incubated with HBSS.

Mitochondrial Isolation

To measure the individual NAD⁺ level in mitochondrial and cytoplasm,Neuro2a cells were seeded in 10 cm² petri dish, then treated with 1 mMNRH for overnight. The cells were harvested with trypsin and pelleted byspinning at 3000x g for 5 min. The mitochondrial fractions were isolatedusing Mitochondrial Isolation Kit for Mammalian Cells (ThermoScientific) according to manufacturer's manual. Protein concentrationswere later measured using Bradford assay for normalization.

Cytotoxicity Test

To test the effect of NRH against different toxins, Neuro2a, HEK293 orINS1 cells were seeded in 6-well plate and grow until confluent. 500 μMhydrogen peroxide, 400 μM MMS, or 100 nM vincristine was added to mediawith or without the co-incubation of 1 mM NRH. Cells were harvestedafter 7 hr with trypsin and used for NAD⁺ measurement. Trypan blue wasused to distinguish dead and live cells during cell count.

Stability Test of NRH

To assess the stability of NRH in different pH environment, 1 mM NRH wasincubated in 150 mM phosphate buffer at pH 6, 7, 7.4, 8, and 9, and wasinjected into EC 250/4.6 Nucleosil 100-5 C18 column on a Hitachi EliteLachrom™ HPLC system equipped with Diode Array Detector L-2450 after 1,4, 7 and 10 hr. The incubation temperature on HPLC has been set to 10°C. The C18 column was eluted with 20 mM ammonium acetate at 1 mL/min for25 min, then with 20 mM ammonium acetate and 20% Methanol for 20 min.NRH was characterized by its peak at 340 nm. The peaks were quantifiedand used for calculation.

Example 9

This example demonstrates the effect of dihydronicotinamide riboside(NRH) on HEK293 and Neuro2A cell counts.

Cells were seeded into tissue culture coated 6-well plates and grown for18 hours in complete media (DMEM, 10% FBS) (HEK293: seeded 0.8 millioncells per well; Neuro2A: seeded 0.6 million cells per well). Cells werethen treated with NRH (1 mM) or equivalent volume of sterile water incomplete media for 7 hours. Media was aspirated and cells were suspendedin fresh complete media and stained with 0.2% trypan blue for countingby hemacytometer. FIGS. 1A and 1B shows the viable (unstained) cellcounts for both HEK293 and Neuro2A cells treated with water or NRH.FIGS. 1C and 1D shows the non-viable (stained) cell counts for bothHEK293 and Neuro2A cells treated with water or NRH. Treatment with NRHdoes not appear to affect cell viability or growth as compared to thewater-only controls. The non-viable cell counts are also reduced,suggesting NRH has cell maintenance effects.

Example 10

This example demonstrates the effect on NAD+ levels in HEK293 cellsresulting from treatment with dihydronicotinamide riboside (NRH) ascompared to treatment with water and with nicotinamide riboside (NR).

HEK293 cells were grown to 100% confluence in tissue culture treated6-well plates in complete media (DMEM, 10% FBS). Cells were treated with100 uM-1 mM NRH for 6 or 16 hours in complete media. Water-only andnicotinamide riboside (NR) control treatments were run. Cells wereharvested and NAD+ levels were measured using the NAD+ cycling assay.NAD⁺ levels are reported in pmol per 10⁶ cells. NAD+ levels resultingfrom a 16 hour treatment of HEK293 cells with water, 100 μM NRH, 500 μMNRH, and 500 μM NR are shown in FIG. 2A. NAD⁺ levels resulting from an 8hour treatment of HEK293 cells with water, 100 μM NRH, 500 μM, 1000 μMRNH, and 1900 μM NR are shown in FIG. 2B. NAD⁺ resulting from a 16 hourtreatment of HEK293 cells with water, 750 μM NRH, and 750 μM NR areshown in FIG. 2C. Across all time points and concentrations, cellstreated with NRH had increased NAD⁺ levels with magnitudes ranging from76% to 176% increase in NAD+ over water-only controls. Untreated cellswere found to have NAD⁺ levels consistent with a previously reportedvalue for HEK293 cells of 320 pmol/10⁶ cells. In all cases NRH had agreater effect on NAD⁺ levels as compared to the NR control.

Example 11

This example demonstrates the effect on NAD⁺ levels in Neuro2A cellsresulting from treatment with dihydronicotinamide riboside (NRH) ascompared to treatment with water and with nicotinamide riboside (NR).

Neuro2A cells were grown to 100% confluence in tissue culture treated6-well plates in complete media (DMEM, 10% FBS). Cells were treated with500 uM NRH for 6 hours in complete media. DMSO-only, water-only and NRcontrol treatments were run. Cells were harvested and NAD+ levels weremeasured using the NAD⁺ cycling assay. NAD⁺ levels are reported in pmolper 10⁶ cells. In FIG. 3A, cells treated with NRH showed a 484% increasein NAD⁺ levels over the untreated control. In FIG. 3B, NRH treatmentgave a 236% increase in NAD⁺ levels. Untreated cells were found to haveNAD⁺ levels consistent with a previously reported value for Neuro2Acells of 680 pmol/10⁶ cells.

Example 12

This example demonstrates the effect of treatment with Neuro2A cellswith hydrogen peroxide and dihydronicotinamide riboside (NRH).

Neuro2A cells were grown to 100% confluence in tissue culture treated6-well plates in complete media (DMEM, 10% FBS). Cells were treated with1000 uM NRH (columns 2 and 4 of FIG. 4 from left to right) or vehicle(columns 1 and 3 of FIG. 4) for 7 hours in complete media. Lanes 3 and 4of FIG. 4 show cells that were cotreated with 500 uM hydrogen peroxide.Cells were harvested and viable cells were counted by haemocytometer.The results are shown in FIG. 4. Statistical significance was achievedbetween columns 1 and 3, and 3 and 4 (p<0.05). This result shows thatNRH can rescue cells subject to a cell killing stress, meant to mimicphysiologically relevant tissue killing stress.

Example 13

This example demonstrates that dihydronicotinamide riboside (NRH)enhances NAD⁺ levels in different cell types

To test if NRH can increase cellular NAD⁺ level among differentmammalian cell types, 1 mM NRH was used to treat neuronal cells(Neuro2a, U87, F98 and LN229), kidney cells (HEK293), muscle cells(C2Cl2) and beta cells (INS1 and MIN6) for 7 hr. NRH has shown robustNAD⁺ enhancing effect in all tested cell lines, especially in Neuro2acells where NAD⁺ level was raised up to around 10 fold comparing tountreated control. The results are set forth in Table 1.

TABLE 1 Percentage NAD+ increase Cell Type Cell Line by 1 mM NRHNeuronal cells Neuro2A 979% ± 63% U87 659% ± 76% F98 355% ± 45% LN229386% ± 90% Kidney cells HEK293 382% ± 48% Muscle cells C2C12 611% ± 40%Beta cells INS1 299% ± 34% MIN6 384% ± 32%

Example 14

This example demonstrates that NRH enhances NAD⁺ levels in atime-dependent and dose-dependent manner.

In order to assess if the impact of NRH on NAD⁺ level is time-dependent,Neuro2a cells were treated with 1 mM NRH for 0, 1 or 7 hr. Within 1 hourNAD⁺ contents increased by 20%, whereas at 7 hours, NRH treatmentincreased by 400% comparing to control, suggesting that the effect ofNRH increasing with incubation time (FIG. 5). Also, to test if NRH hasdose-dependent effect, Neuro2a cells were incubated with NRH between 10to 1000 μM for 7 hour. The cellular NAD⁺ levels were raised by 121% with10 μM NRH and escalated gradually as the treatment concentrationincreased (FIG. 6). These results were further confirmed by HPLC showinghigher NAD⁺ peak in cell lysates from NAD⁺ treated cells. These datasuggest the effect of NRH in Neuro2a cells are both time anddose-dependent.

Example 15

This example demonstrates that NRH has a dose-dependent NAD⁺ enhancingeffect in INS1 cells.

The dose-dependent effect of NRH was tested in INS1 cells for 7 hours.NAD⁺ levels were gradually increased by elevating NRH concentration inthe treatment. The effect of NRH was compared with 1 mM NR. 100 μM NRHexerted similar NAD⁺ increase as 1 mM NR treatment (FIG. 7), showingthat NRH was a stronger NAD⁺ enhancer comparing to NR in INS1 cells.

Example 16

This example demonstrates that NRH has dose-dependent NAD⁺ enhancingeffect in primary neurons.

To test if NRH has similar effect in primary cells as immortalized celllines, primary neurons cells harvested from rat brains were treated with100 μM to 1 mM NRH for 6 hr. The primary neurons respond to 100 μM NRHtreatment with 4.9 fold increase in their NAD⁺ levels but the NAD⁺levels maximized with 500 μM NRH treatment, by which NAD⁺ were increasedby around 8 fold, as shown in FIG. 8.

Example 17

This example demonstrates that NRH elevated NAD⁺ level in bothmitochondria and cytoplasm.

It was evaluated if the NAD⁺ enhancing effect of NRH is restricted tocell compartment. Neuro2a cells were treated with 1 mM NRH overnight andtheir mitochondria and cytoplasm were separated for NAD⁺ analyses. NRHelevated the NAD⁺ level not only in cytoplasm but also in mitochondria,as shown in FIG. 9, suggesting NRH may have potential benefits insupporting mitochondrial biogenesis by supplying NAD⁺ directly tomitochondria.

Example 18

This example demonstrates that NRH is stable in alkaline buffer.

To further understand the chemical properties of NRH, NRH was incubatedphosphate buffers pHed to 6, 7, 7.4, 8, and 9. HPLC chromatograph showedthat NRH was rapidly degraded in slightly acidic buffer at pH 6. In aneutral pH, at pH 7 and pH 7.4, NRH was not very stable and degraded by35% and 26% after 10 hr incubation at 10° C. NRH appears to be morestable at slightly alkaline conditions, in which 84% of NRH was remainedat pH 8 and 94% of NRH was preserved at pH 9 after 10 hr as shown inFIG. 10. Therefore, NRH is more stable in slightly alkaline conditioncomparing to neutral or acidic environment.

Example 19

This example demonstrates that NRH protected cells from cytotoxicity byenhancing NAD⁺ content.

In order to evaluate if the NAD⁺ enhancing effect of NRH can protectcells from oxidative stress and DNA damage-induced cytotoxicity, Neuro2acells were treated with hydrogen peroxide or methyl methanesulfonate(MMS) with or without the presence of NRH. Both hydrogen peroxide andMMS induced significant cell death and NAD⁺ depletion, but the additionof NRH preserved cellular NAD⁺ content and protected the cells fromcytotoxicity (FIG. 11A, FIG. 12A). Similar protection from NRH was alsoobserved in INS1 cells (FIG. 11B) and HEK293 cells (FIG. 12B). Also,when stressed with a neurotoxin, vincristine, Neuro2a cells alsosuffered from cell death and NAD⁺ deprivation, whereas NRH restored NAD⁺level in cells and maintained living cell count (FIG. 13).

Example 20

This example demonstrates that NRH preserved insulin-secretion capacityin beta cells.

To examine if NRH can preserve the normal insulin secreting function inbeta cells under oxidative stress, hydrogen peroxide and NRH were addedto INS1 cells in glucose-induced insulin secretion assay. NRH restoredthe NAD⁺ level depleted by hydrogen peroxide treatment, even to a higherextent comparing to NR treatment as shown in FIG. 14A. When assessingthe insulin secretion ability of INS1 cells, high glucose media induceda significantly increased insulin release into the media and the insulinsecretion was largely sabotaged by hydrogen peroxide treatment. Additionof either NRH or NR has restored the insulin secretion level back to asimilar extent as untreated control as shown in FIG. 14B, demonstratingboth NRH and NR can protect the insulin secreting function of INS1 cellsunder oxidative stress induced by hydrogen peroxide.

Example 21

This example demonstrates that cellular NAD⁺ concentration is increasedin HEK cells by treatment with a dihydronicotinate riboside compound.

HEK cells were plated in 6-well plates until 90% confluent. Cells weretreated with ethyl dihydronicotinate riboside (Et NaR-H, i.e.ethyl-1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-dihydropyridine-3-carboxylate)(50 μM, 100 μM and 500 μM), propyl dihydronicotinate riboside (Pr NaR-H,i.e. propyl-1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-dihydropyridine-3-carboxylate)(50 μM, 100 μM and 500 μM), or dihydronicotinamide riboside (NHR) (50 μMand 500 μM) as a positive control. After 6 hours of incubation, cellswere harvested with Trypsin. A cell count was obtained from each cellsuspension by hemacytometer. The cell pellets were used for NAD⁺measurement. As shown in FIG. 15, cellular NAD concentration isincreased by treatment with dihydronicotinate riboside compounds.

Example 22

This example demonstrates that cellular NAD⁺ concentration is increasedin HEK cells by treatment with a dihydronicotinate riboside compound.

HEK cells were plated in 6-well plates until 90% confluent. Cells weretreated with butyl dihydronicotinate riboside (Bu NaR-H, i.e.butyl-1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-dihydropyridine-3-carboxylate)(50 μM), heptyl dihydronicotinate riboside (Hep NaR-H, i.e.heptyl-1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-dihydropyridine-3-carboxylate)(50 μM), octyl dihydronicotinate riboside (Oct NaR-H, i.e.octyl-1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-dihydropyridine-3-carboxylate)(50 μM) or dihydronicotinamide riboside (NHR) (50 μM) as a positivecontrol. After 6 hours of incubation, cells were harvested with Trypsin.A cell count was obtained from each cell suspension by hemacytometer.The cell pellets were used for NAD⁺ measurement. The average NAD⁺ wasdetermined and the percentages of average NAD⁺ as compared to controlare set forth in Table 2. As is apparent from the results set forth inTable 2, cellular NAD concentration is increased by treatment withdihydronicotinate riboside compounds.

TABLE 2 Compound Average NAD⁺ (pmoles/10⁶ cells) NRH 145% Bu NaR—H 124%Hep NaR—H 124% Oct NaR—H 112%

Example 23

This example demonstrates the calculated octanol-water partitioncoefficients (c Log P) of compounds in accordance with an embodiment ofthe invention.

The octanol-water partition coefficients of compounds were calculatedusing the ChemDraw™ program version 12.0. The molecular weights andcalculated c Log P values are set forth in Table 3.

TABLE 3

X MW CLogP MW CLogP NH₂ 255 −5.1 256 −1.99 OH 256 −4.19 257 −1.25 MeO270 −4.42 271 −0.73 EtO 284 −3.89 285 −0.20 n-PrO 298 −3.37 299 0.33i-PrO 298 −3.59 299 0.11 n-BuO 312 −2.84 313 0.86 n-PeO 326 −2.31 3271.38 n-HexO 340 −1.78 341 1.91 n-HepO 354 −1.25 355 2.44 n-OctO 368−0.72 369 2.97

As is apparent from the results set forth in Table 3, thedihydronicotinoyl riboside esters have octanol-water partitioncoefficients that are significantly higher than the octanol-waterpartition coefficients of the nicotinoyl riboside esters, indicatingthat the reduced compounds have significantly higher lipophilicity thanthe non-reduced compounds.

Example 24

This example demonstrates the effect of administration of NRH on NAD+levels in whole blood in mice.

4 C57/B6 mice (mass ranges 25-30 g each) were injected intraperitoneallywith 1000 mg/kg NRH with NRH dissolved in 100 microliter sterilephosphate buffered saline and 4 of the same littermates were injectedthe same volume of sterile phosphate buffered saline. At 2 and 4 hoursblood was drawn by capillary and samples were assayed by known assaymethods to assess for NAD content. The results are shown in FIG. 16. Asis apparent from FIG. 16, NRH-treated animals have higher NAD levels at2 and 4 hours in whole blood as compared with control animals injectedat the same time with phosphate buffered saline.

Example 25

This example demonstrates the effect on NAD⁺ levels in HEK293 cellsresulting from treatment with dihydronicotinamide riboside (NRH) ascompared to treatment with water and with nicotinamide riboside (NR).

HEK293 cells were treated with 1 mM NRH and with 1 mM NR as described inExample 11. NAD⁺ cells were determined as compared to control and theresults set forth in

TABLE 4 Compound Average NAD⁺ (pmoles/10⁶ cells) NRH (1000 mM) 236% NR(1000 mM) 148%

As is apparent from the results set forth in Table 4, treatment ofHEK293 cells with 1000 mM of NRH resulted in a 236% increase in NAD⁺,while treatment of HEL293 cells with 1000 mM of NR resulted in a 148%increase in NAD⁺.

The invention is exemplified by the following embodiments:

1. A compound of formula (I):

wherein R¹, R², and R³ are independently selected from hydrogen, formyl,optionally substituted C₁-C₁₂ alkylcarbonyl, and C₆-C₁₀ optionallysubstituted arylcarbonyl,

X is NHR⁴, NR⁴R⁵, or OR⁶,

R⁴ and R⁵ are independently optionally substituted C₁-C₁₂ alkyl oroptionally substituted C₆-C₁₀ aryl, and

R⁶ is hydrogen, optionally substituted C₁-C₁₂ alkyl, or optionallysubstituted C₆-C₁₀ aryl,

wherein the alkyl or aryl portion of R⁴-R⁶ is optionally substitutedwith one or more substituents selected from halo, amino, alkyl, alkoxy,aryloxy, hydroxyalkyl, and any combination thereof,

with the provisos that, when X is OR⁶ and R⁶ is hydrogen, R¹, R², and R³are not all hydrogen or acetyl,

when X is OR⁶ and R⁶ is hydrogen, R¹ is not benzoyl and R² and R³ arenot hydrogen, and

when X is OR⁶ and R⁶ is methyl or ethyl, R¹, R², and R³ are not allacetyl,

or a salt thereof.

2. The compound of embodiment 1, wherein X is OR⁶, R⁶ is optionallysubstituted C₁-C₁₂ alkyl, and R¹, R², and R³ are hydrogen.

3. The compound of embodiment 1 or 2, wherein the compound is selectedfrom:

4. The compound of embodiment 1, wherein X is OR⁶, R⁶ is optionallysubstituted C₁-C₁₂ alkyl, and R¹, R², and R³ are C₁-C₁₂ alkylcarbonyl.

5. The compound of embodiment 1, wherein X is NHR⁴

6. The compound of embodiment 1, wherein X is NR⁴R⁵.

7. The compound of embodiment 5 or 6, wherein R¹, R², and R³ arehydrogen.

8. The compound of embodiment 5 or 6, wherein R¹, R², and R³ are C₁-C₁₂alkylcarbonyl.

9. A pharmaceutical composition comprising a compound of any one ofembodiments 1-8 or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.

10. A dietary supplement or food ingredient composition comprising acompound of any one of embodiments 1-8 or a salt thereof.

11. A process for the preparation of a compound of formula (II):

wherein R¹, R², and R³ are independently selected from hydrogen, formyl,optionally substituted C₁-C₁₂ alkylcarbonyl, and C₆-C₁₀ optionallysubstituted arylcarbonyl, and

R⁶ is hydrogen, optionally substituted C₁-C₁₂ alkyl, or optionallysubstituted C₆-C₁₀ aryl,

wherein the alkyl or aryl portion of R⁶ is optionally substituted withone or more substituents selected from halo, amino, alkyl, alkoxy,aryloxy, hydroxyalkyl, and any combination thereof,

wherein the process comprises the steps of:

(i) providing a compound of formula (III):

(ii) reducing the compound of formula (III) with a reducing agent toprovide the compound of formula (II), and

(iii) isolating the compound of formula (II).

12. The process of embodiment 11, wherein the reducing agent is sodiumdithionate.

13. The process of embodiment 11, wherein the compound of formula (III)is isolated by chromatography.

14. A method for increasing cell NAD⁺ production comprisingadministering to a cell a compound of any one of embodiments 1-8 or asalt thereof.

15. The method of embodiment 14, wherein the cell is in a mammal havinga lipid disorder, a metabolic dysfunction, a cardiovascular disease, CNSor PNS trauma, a neurodegenerative disease or condition, or hearingloss, or is in a mammal that has been exposed to a toxic agent.

16. The method of embodiment 14, wherein the cell is in a mammal at riskfor hearing loss.

17. The method of embodiment 14, wherein the cell is in a mammal,wherein the compound is administered in an amount effective forpromoting the function of the metabolic system, promoting musclefunction or recovery, promoting the function of the auditory system, orpromoting cognitive function.

18. A method of improving mitochondrial densities in a cell, wherein themethod comprises administering to the cell a compound of any one ofembodiments 1-8 or a salt thereof.

19. The method of embodiment 18, wherein the cell is in a mammal havinga lipid disorder, a metabolic dysfunction, a cardiovascular disease, CNSor PNS trauma, a neurodegenerative disease or condition, hearing loss,or is in a mammal that has been exposed to a toxic agent.

20. The method of embodiment 18, wherein the cell is in a mammal at riskfor hearing loss.

21. The method of embodiment 18, wherein the cell is in a mammal,wherein the compound is administered in an amount effective forpromoting the function of the metabolic system, promoting musclefunction or recovery, promoting the function of the auditory system, orpromoting cognitive function.

22. A method for increasing mammalian cell and tissue NAD⁺ productioncomprising administering to a cell a compound of any one of claims 1-8or a salt thereof.

23. The method of embodiment 22, wherein the disease or conditionresults from toxic effects of high fat diets.

24. A method of treating or preventing a disease or condition in amammal in need thereof, wherein the method comprises administering tothe mammal a compound of any one of embodiments 1-8 or a salt thereof,wherein the disease or condition is neurodegeneration caused byAlzheimer's disease.

25. A method of improving mitochondrial densities, insulin sensitivity,or exercise endurance in a mammal, wherein the method comprisesadministering to the mammal a compound of any one of embodiments 1-8 ora salt thereof.

26. A method of protecting a mammal from neurotrauma, wherein the methodcomprises administering to the mammal a compound of any one ofembodiments 1-8 or a salt thereof.

27. The method of embodiment 26, wherein neurotrauma results from blastinjury or noise.

27. A method of reducing toxicity induced by a HMGCoA reductaseinhibitor in a mammal, which method comprises administering to themammal a therapeutically effective amount of a compound of any one ofembodiments 1-8 or a salt thereof,

wherein the mammal has been administered the HMGCoA reductase inhibitorin an amount that produces toxicity in the mammal in the absence of theadministration of the compound of formula (I), and

wherein the administration of the compound of embodiment 1 reduces thetoxicity in the mammal.

28. A method of reducing toxicity induced by a HMGCoA reductaseinhibitor in a mammal, which method comprises administering to themammal a therapeutically effective amount of a compound of any one ofembodiments 1-8 or a salt thereof and then administering to the mammalthe HMGCoA reductase inhibitor in an amount that produces toxicity inthe mammal in the absence of the administration of the compound offormula (I), whereby toxicity that would have been induced by the HMGCoAreductase inhibitor is reduced in the mammal by the administration ofthe compound of any one of claims 1-8.

29. A method of reducing toxicity induced by a HMGCoA reductaseinhibitor in a mammal, which method comprises administering to themammal a therapeutically effective amount of a compound of any one ofembodiments 1-8 or a salt thereof, whereby toxicity induced by theHMGCoA inhibitor is reduced in the mammal, wherein the compound offormula (I) is administered to the mammal following manifestation oftoxicity by the mammal.

30. A method of reducing toxicity induced by a genotoxic agent in amammal, which method comprises administering to the mammal atherapeutically effective amount of a compound of any one of embodiments1-8 or a salt thereof, wherein the mammal has been administered thegenotoxic agent in an amount that produces toxicity in the mammal in theabsence of the administration of the compound of formula (I), andwherein the administration of the compound reduces the toxicity in themammal.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method for increasing cell NAD⁺production or for improving mitochondrial densities in a cell consistingof administering to a cell a compound of formula (I):

wherein R¹, R², and R³ are each hydrogen, and X is NH₂, or a saltthereof, optionally in combination with a multi-vitamin, a mineral, anacceptable carrier, or any combination thereof.
 2. The method of claim1, wherein the method increases cell NAD+ production.
 3. The method ofclaim 2, wherein the cell is in a mammal having a lipid disorder, ametabolic dysfunction, a cardiovascular disease, CNS or PNS trauma, aneurodegenerative disease or condition, or hearing loss, or is in amammal that has been exposed to a toxic agent.
 4. The method of claim 2,wherein the cell is in a mammal that has hearing loss.
 5. The method ofclaim 2, wherein the cell is in a mammal, wherein the compound isadministered in an amount effective for promoting the function of themetabolic system, promoting muscle function or recovery, promoting thefunction of the auditory system, or promoting cognitive function.
 6. Themethod of claim 1, wherein the method improves mitochondrial densitiesin the cell.
 7. The method of claim 6, wherein the cell is in a mammalhaving a lipid disorder, a metabolic dysfunction, a cardiovasculardisease, CNS or PNS trauma, a neurodegenerative disease or condition,hearing loss, or is in a mammal that has been exposed to a toxic agent.8. The method of claim 6, wherein the cell is in a mammal that hashearing loss.
 9. The method of claim 6, wherein the cell is in a mammal,wherein the compound is administered in an amount effective forpromoting the function of the metabolic system, promoting musclefunction or recovery, promoting the function of the auditory system, orpromoting cognitive function.
 10. A dietary supplement or foodingredient composition comprising the compound of claim 1 or a saltthereof, wherein the dietary supplement or food ingredient compositionis a solid composition suitable for oral administration comprising thecompound or salt and an acceptable carrier.
 11. A method for increasingNAD⁺ concentration in a mammal consisting of administering to the mammala compound of formula (I):

wherein R¹, R², and R³ are each hydrogen, and X is NH₂, or a saltthereof, optionally in combination with a multi-vitamin, a mineral, anacceptable carrier, or any combination thereof.
 12. The method of claim11, wherein the administration increases NAD⁺ concentration in blood ofthe mammal.
 13. The method of claim 11, wherein the administrationincreases NAD⁺ concentration in a tissue of the mammal.